Method and composition for treatment of skeletal dysplasias

ABSTRACT

The present invention relates to a method for the treatment of skeletal dysplasia by administering to a patient a composition comprising a therapeutically effective amount of at least one C-type natriuretic peptide (CNP).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 11/840,631filed Aug. 17, 2007, which is a division of U.S. application Ser. No.10/664,605 filed Sep. 15, 2003, U.S. Pat. No. 7,276,481, which is acontinuation of the U.S. national phase of International applicationPCT/IL02/00229 filed Mar. 20, 2002, which claims the benefit of U.S.application No. 60/276,939 filed Mar. 20, 2001. The content of eachprior application is expressly incorporated herein by reference thereto.

FIELD OF THE INVENTION

The present invention relates to compositions for treatment of skeletaldysplasias, such as achondroplasia, and in particular to pharmaceuticalcompositions for bone elongation in disorders related to abnormal bonegrowth comprising natriuretic peptides or natriuretic factors.

BACKGROUND OF THE INVENTION Bone Development

Endochondral ossification is a fundamental mechanism for bone formation,whereby cartilage is replaced by bone. Endochondral ossificationrequires the sequential formation and degradation of cartilaginousstructures that serve as molds for the developing bones. The process ofendochondral ossification in the cartilaginous growth plate, which islocated at both ends of vertebrae and long bones, determineslongitudinal bone growth.

During fetal life and until the end of puberty, longitudinal bone growthtakes place via endochondral ossification of the growth plate located atthe epiphyses (ends) of long bones. The growth plate is divided intoseveral zones of cartilage forming cells, or chondrocytes, with distinctpatterns of gene expression. In the Reserve Zone, cells are small andrelatively inactive. In the adjacent Proliferative Zone, chondrocytesproliferate, arrange themselves in columns and eventually undergohypertrophy. In the lower Hypertrophic Region towards the cartilage-bonejunction, cells are big and highly active but exhibit no further celldivision. The matrix surrounding the hypertrophic cells calcifies andthe lowermost cells undergo programmed cell death. Cell death isaccompanied by the removal of the cartilaginous matrix and itsreplacement by bone through the concerted action of recruited bonecells, namely osteoclasts and osteoblasts.

Signaling Pathways in Bone Development

The process of endochondral ossification is the result of the concertedaction of several signaling pathways. The signaling pathway triggered byactivation of Fibroblast Growth Factor (FGF) receptors have been shownto be involved in several stages of limb and bone development. A numberof birth defects are associated with mutations in the genes encoding FGFreceptors (FGFR). For example a mutation in FGFR1 is associated withPfeiffer syndrome. Certain other mutations in FGFR2 are associated withCrouzon, Pfeiffer, Jackson-Weiss, Apert or Beare-Stevenson syndromes.The clinical manifestation of Apert syndrome (AS) is characterized byboth bony and cutaneous fusion of digits of the hands and the feet.Broad thumbs and halluces distinguish Pfeiffer syndrome, while inCrouzon syndrome limbs are normal but a high degree of proptosis isevident. The most prominent malformation syndrome associated with thesemutations is craniosynostosis (the premature fusion of the skull bonessutures).

FGFR3 has an inhibitory role in bone elongation as demonstrated by thefact that mice lacking this receptor exhibit a phenotype of skeletalovergrowth. Moreover, mutations at various positions in this receptorresult in skeletal dysplasias (SD). Thanatophoric dysplasia is a severeand lethal form, while hypochondroplasia is a milder form thanAchondroplasia. Examination of the sequence of FGFR3 in Achondroplasiapatients identified a mutation in the transmembrane domain of thereceptor (reviewed in Vajo et al. (2000) Endocrine Rev 21:23-39).

Achondroplasia is the most common form of short-limbed dwarfismoccurring with a frequency of 1:20,000 live births. Patients showcharacteristic shortening of proximal long bones (rhizomelia), relativemacrocephaly, depressed nasal bridge and lumbar lordosis.

Achondroplasia is mainly caused by a Gly380Arg (G380R) mutation in thetransmembrane domain of the FGFR3 and is transmitted in an autosomaldominant fashion (Shiang et al. (1994) Cell 78: 335-342 and Rousseau etal. (1994) Nature 371: 252-254). A Gly375Cys (G375C) mutation has alsobeen reported in some Achondroplasia patients. These mutations affectthe process of endochondral ossification by inhibiting proliferation anddelaying maturation of chondrocytes in the growth plate cartilage oflong bones, resulting in decreased elongation.

Other major regulators of bone growth include growth hormone (GH,reviewed in Kelly et al., (2001) Horm Res 55 Suppl 2:14-7); insulin-likegrowth factor 1 (IGF-1, reviewed in McCarthy and Centrella (2001) GrowthHorm IGF Res 11:213-9), glucocorticoids (GC) thyroid hormone (TH, Harveyet al., (2002) Mol Genet Metab 75:17-30) and Vitamin D (van Leeuwen etal, (2001) Steroids 66:375-80).

Each of these molecules exerts its function by binding to specificcell-surface or nuclear receptors of skeletal cells.

Natriuretic Peptides

Natriuretic peptides are known for their role in cardiovascularhomeostasis, diuresis, natriuresis and vasodilation. Four isoformsconstitute this family: atrial natriuretic peptide (ANP), brainnatriuretic peptide (BNP), C-type natriuretic peptide (CNP) anddendroaspis natriuretic peptide (DNP). While ANP and BNP are circulatingpeptides produced by the atria and the ventricle respectively, CNP ishardly found in circulation and is mainly produced in the brain, invascular endothelial cells and other tissues where it is supposed towork in an autocrine/paracrine manner (Chen and Burnett (1998) J.Cardiovasc. Pharm. 32 Suppl 3:S22-8). DNP is present in human plasma andatrial myocardium (Chen et al (2000) Curr Cardiol 2:198-205) and itssequence disclosed (Schweltz et al (1992) JBC 267:13928-32). CNP fromdifferent species have been disclosed in U.S. Pat. Nos. 5,336,759(frog); 5,338,759 (chicken); 5,973,134 (rat); 6,020,168 (pig) and6,034,231 (human).

Natriuretic peptides effect their biological role through two receptors:NPR-A and NPR-B. These receptors have cytoplasmic guanylyl cyclasedomains, which are activated upon ligand binding and lead toaccumulation of intracellular cGMP. Some of the effects of cGMP aremediated through two known protein kinases: cGMP-dependent proteinkinase I and II. The peptides bind the receptors with differentaffinities: ANP≧BNP>>CNP for NPR-A and CNP>ANP≧BNP for NPR-B. The tissuedistribution of each receptor is different. While NPR-A is expressed invasculature, kidney and adrenal glands, NPR-B is mainly expressed in thebrain.

NPR-C, a third receptor devoid of the kinase and cytoplasmic GC domainsis generally considered to be a clearance receptor for removingnatriuretic peptides from the circulation, though some other biologicalfunctions have been attributed to it (Murthy and Makhlouf (1999) JBC274:17587-92). This is a widely distributed receptor expressed in almostall the tissues that express a guanylyl cyclase receptor. U.S. Pat. No.5,846,932 discloses potent ANP variants having decreased affinity forthe human clearance or C-receptor. These ANP variants exhibitnatriuretic, diuretic and vasorelaxant activity but have increasedmetabolic stability, making them suitable for treating congestive heartfailure, acute kidney failure and renal hypertension. Furthermore,WO00/61631 discloses novel pentapeptide antagonists of the NPR-C.

Natriuretic peptides have a short half life in vivo. In addition to theclearance receptor, they are further cleared from the circulation bydegradation. The peptides are cleaved at specific sites, by the neutralendopeptidase 24.11 (NEP) which is found in endothelial cells coveringthe vascular walls. Human BNP is more resistant to this degradationwhile ANP and CNP are readily degraded by this enzyme. Inhibition of NEPby inhibitors, including the compounds thiorphan or candoxatril(Ohbayashi et al. (1997) Clin. Exp. Pharma. Physiol. 25: 986-91; Brandtet al. (1997) Hyperten. 30: 184-90), increases the concentration ofendogenous or administered peptides in the circulation.

CNP, like ANP, BNP and DNP, was shown to exhibit natriuretic andhypotensive actions. Novel CNP-related peptides capable of eliciting astrong cGMP response and suppressing the growth of vascular smoothmuscle cells have been disclosed in U.S. Pat. No. 5,434,133. Alsodisclosed are the amino acids responsible for the cGMP producingactivities and novel CNP variants capable of inhibiting abnormal growthof smooth muscle cells, for the treatment of, inter alia, restenosis andarteriosclerosis.

Transgenic mice, over-expressing BNP show a skeletal phenotypecharacterized by overgrowth of the axial and appendicular skeleton (Sudaet al. (1998) PNAS 95: 2337-42). Moreover, mice that are null mutantsfor the clearance receptor, NPR-C, exhibit similar skeletal overgrowth,consistent with a role for the local modulation of natriuretic peptideslevels by NPR-C (Matsukawa et al. (1999) PNAS 96: 7403-08). CNP and itsspecific receptor, NPR-B, have been shown to be expressed in theproliferating zone of the growth plate in fetal mouse tibia while NPR-Chas been shown to be expressed in the region of hypertrophicchondrocytes and in osteoblasts (Yamashita et al. (2000) J Biochem 127:177-9). After the date of the present invention, Chuso et al (Chusho etal. (2001) PNAS 98:4016-21) have disclosed CNP knockout mice whichexhibit skeletal phenotypes histologically similar to those seen inAchondroplasia mice. They also reveal the rescue of the CNP knock outskeletal defects by tissue-specific ectopic CNP expression in the growthplate. Moreover, ex vivo experiments (fetal bone organ culture) fromwild type animals have shown that CNP, more than BNP and ANP, can inducebone elongation (Yasoda et al. (1998) JBC 273: 11695-700, Mericq et al.(2000) Ped Res 47: 189-93).

While much is known about the components of signaling pathways thatcontribute to the process of endochondral ossification, little is knownabout the complex interactions between them that coordinate longitudinalbone growth.

SUMMARY OF THE INVENTION

The present invention sets out to provide a method for the treatment ofskeletal dysplasias. The present invention also provides pharmaceuticalcompositions useful in the treatment of skeletal dysplasias. Yet novelcompounds and compositions useful in the methods of the invention arealso provided.

The method and composition of the invention affect bone elongation,inter alia, by increasing the size of the growth plate of the bone,specifically of limb bones, in skeletal dysplasias such asachondroplasia.

The present invention provides a pharmaceutical composition comprisingnatriuretic peptides (NP) or functional variants useful in effectingbone elongation and treating skeletal dysplasias.

The present invention also provides NP variants with increasedstability.

The present invention further provides a method to enhance NPstabilization in circulation.

Still, the present invention provides a method of delivering NP or itsvariants to a target site.

The invention provides the methods of effecting bone elongation andtreating skeletal dysplasias which comprise using natriuretic peptides(NP), their conjugates, variants and derivatives in.

The method of the invention for treating skeletal dysplasias includesthe step of administering to a patient an effective amount of an NP. Inone currently preferred embodiment of the present invention thenatriuretic peptide is CNP. In another currently preferred embodimentthe natriuretic peptide is a CNP variant. The method may further includea step of administering to the patient an inhibitor of neutralendopeptidase 24.11 (NEP). Suitable compounds for inhibiting NEP areknown in the art, including but not limited to thiorphan or candoxatril.Administration of such an inhibitor of neutral endopeptidase may beperformed either separately or simultaneously with the administration ofNP. It may also include administering a clearance receptor (NPR-C)inhibitor either alone or in conjunction with administration of NP.

Hyperactivation of the FGFR pathways has been implicated in several boneskeletal dysplasias. In particular, over stimulation of FGFR3 results inbone growth inhibition. The method of the invention for treatingskeletal dysplasias includes administering to a patient a pharmaceuticalcomposition comprising an NP and a receptor kinase inhibitor, inparticular a tyrosine kinase inhibitor including, but not limited to,those disclosed in U.S. Pat. No. 6,329,375 or 6,344,459.

In another currently preferred embodiment NP is targeted to a desiredtissue, specifically the growth plate of the bones. This may be achievedby methods known to one skilled in the art and include, in a nonlimitingmanner, a chimeric protein comprising an NP fused to a carrier domain toform a fusion protein. The NP includes all forms of the NPs, CNP andderivatives or variants. A carrier domain includes, for example, ahormone or a ligand for a receptor expressed in the target tissue. Thecarrier protein may be an active agonist or an inactive targeting moietyor a variant or mutant thereof. According to one currently preferredembodiment CNP or a functional variant is fused to growth hormone. Analternative embodiment comprises conjugating at least one NP to acarrier protein to form an NP-carrier protein conjugate.

Alternatively, NP may be conjugated to an agent to prolong its half lifein circulation or to a peptide that facilitates translocation across acell membrane.

Administration of NP to a patient can be achieved by any suitable routeof administration, including but not limited to injecting NP to thepatient, inhalation, or implantation of a depot into the patient. Thedepot is preferably implanted at the site of the lesion, the lesionbeing an abnormal bone or a dysplasic bone. The NP may further beadministered by an osmotic pump, such as an Alzet pump. The osmotic pumpcan be implanted subcutaneously, or at any other appropriate site.Preferred sites may be close to the target site of action namely inproximity to the long bones of the limbs, and in particular near theepiphyses.

A further method of administration may be implantation of NP secretingcells. According to one currently preferred embodiment of the inventionthe NP is CNP. The NP may be natural or a variant or analog. The methodsof implanting or transplanting living cells to provide therapeuticallyuseful substances (cell therapy) is known in the art. In one form ofcell therapy, the cells that are implanted have been geneticallymodified in vitro with exogenous genetic material so as to enable thecells to produce a desired biological substance that is useful as atherapeutic agent. Methods of genetically engineering cells are known tothose skilled in the art. Methods for implantation or transplantation ofNP secreting cells include encapsulation of the cells in anyimmunologically inert matrix including gelatin or polymers. According toone currently preferred embodiment the matrix is analginate-polylysine-alginate (APA) complex encapsulating the cells.These methods of administration and other known methods may be utilizedalone or in combination for treating skeletal dysplasia.

The present invention further provides a composition for treatment ofskeletal dysplasias, such as achondroplasia. The composition includes anNP or NP variant and any pharmaceutically acceptable diluent or carrierthereof. According to one currently preferred embodiment of theinvention, the natriuretic peptide is a BNP. According to one currentlymore preferred embodiment of the invention the natriuretic peptide is aCNP. According to one currently most preferred embodiment of theinvention the natriuretic peptide is a CNP variant. The composition mayfurther include any substance, molecule or vehicle capable of increasingthe NP stability in vivo. For example the composition may include aninhibitor of neutral endopeptidase 24.11 (NEP), including but notlimited to thiorphan or candoxatril. In another embodiment thecomposition may include an inhibitor of the natriuretic peptidereceptor-C (NPR-C).

The composition may be in any form suitable for being administered to apatient, including but not limited to in dry, liquid or suspension formor injectable, implantable or transplantable form.

Also provided is a composition for treatment of skeletal dysplasias thatincludes NP secreting cells encapsulated within an immunologically inertmatrix. One currently preferred embodiment includes NP secreting cellsencapsulated in an alginate-polylysine-alginate (APA) complex and asuitable carrier thereof.

According to one currently preferred embodiment of the present inventionnatriuretic peptides and variants thereof are provided for the treatmentof skeletal dysplasias. In another currently preferred embodiment of thepresent invention the NP is CNP and novel CNP variants. The peptidevariants have been modified by removing the five N-terminal amino acidsof CNP, considered to be a part of the ectocyclic domain and havefurther been modified by amino acid substitutions. According to onecurrently more preferred embodiment of the present invention the variantCNP comprises 17 amino acids, from Cysteine5 to the Cysteine22 asdepicted in FIG. 3B, and is identified herein as SEQ ID NO:2. Accordingto one currently most preferred embodiment of the present invention thevariant CNP comprises 17 amino acids and an amino acid substitution at acleavage site, and is depicted as SEQ ID NO:5

Cys-Phe-Gly-Xaa-Xbb-Xcc-Asp-Arg-Ile-Gly-Xdd-Xee-Ser-Xff-Xgg-Gly-Cyswherein

Xaa=Leu, Ile, Val; Xbb=Lys, Leu, Met; Xcc=Leu, Ile, Ala, Val; Xdd=Ser,Ala, Gly, Thr, Asn; Xee=Met, Ala, Lys, Trp; Xff=Gly, Lys, Ala, Leu;Xgg=Leu, Met.

Accordingly, one currently preferred embodiment of the present inventionis the variant having SEQ ID NO: 10 wherein Met17 has been substitutedwith various amino acids and exhibits high cGMP activity. A currentlypreferred embodiment of the present invention provides a CNP variantcomprising a Met17 substitution.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be understood and appreciated more fully fromthe following detailed description taken in conjunction with thedrawings in which:

FIGS. 1A and 1B show the growth rate curves for CNP treated Achheterozygote femora in the ex vivo experiment. FIG. 1A shows the growthrate during the first three days of culture. FIG. 1B shows the growthrate during days 3-15.

FIGS. 2A and 2B show growth rates of femora ex vivo. FIG. 2A depicts theinterpolation of the growth rate curves of FIG. 1. FIG. 2B shows thegrowth rates of CNP or vehicle treated femora derived from wild type,Ach heterozygotes and Ach homozygotes.

FIGS. 3A-C show the amino acid sequences of the NPs and the NEP cleavagesites for CNP. The endocyclic sequences are underlined. The amino acidsare represented by either the one-letter code or three-letter codesaccording to IUPAC conventions. FIG. 3A shows the native human CNP 1-22peptide (SEQ ID NO:1) with the NEP cleavage sites marked with an arrow.FIG. 3B shows the amino acid sequence of CNP-5-22 (SEQ ID NO:2). FIG. 3Cshows the alignment of human ANP, BNP and CNP peptides (SEQ ID NOs: 3, 4and 1, respectively) and the amino acid sequence of the variants of thepresent invention (SEQ ID NO:5).

FIG. 4 shows a scheme of the growth hormone-CNP constructs for producingGH-CNP fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

Achondroplasia is characterized, inter alia, by shortening of proximallong bones. In humans the bone growth plate is active until puberty andbone growth is thus achieved until puberty. Thus, treatment aimed atbone elongation, for example, by increasing the size of limb bone growthplate, would be advantageous during this period.

Treatment of skeletal dysplasias such as achondroplasia, includestreating a shortened bone with NP. The bone may be treated byadministering to a patient an effective amount of NP. The amount of theactive ingredient administered will be determined by the attendingphysician and is generally proportional to the patient's weight.

According to the present invention it is now disclosed that NP caninduce bone elongation in situations of abnormal bone growth such asthose typical of skeletal dysplasias.

The role and use of NPs in bone elongation in situations of abnormalbone growth and in the treatment of skeletal dysplasias, such asachondroplasia, is demonstrated in the following examples andexperiments.

For convenience certain terms employed in the specification, examplesand claims are described here.

The term “natriuretic peptides” or “NP” as referred to herein and in theclaims relates to any of the three isoforms, atrial natriuretic peptide(ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP)and dendroaspis natriuretic peptide (DNP) and to any functional variantsthereof. The NP may be of any species but is preferably human.

The term “FGFR” as used herein denotes a receptor specific for FGF whichis necessary for transducing the signal exerted by FGF to the cellinterior, typically comprising an extracellular ligand-binding domain, asingle transmembrane helix, and a cytoplasmic domain that contains atyrosine kinase activity.

A “ligand” as used herein is a molecule capable of binding a receptor ora receptor analog. The ligands of the hGh-R (human growth hormonereceptor) and FGFRs are the hGH and FGF molecules or variants thereof.The molecule may be chemically synthesized, synthesized by recombinanttechniques or may occur in nature.

The terms “variant”, “derivative” or “mutant” as used hereininterchangeably refer to a polypeptide sequence that possesses somemodified structural property of the native sequence. For example, thevariant may be truncated at either the amino or carboxy termini (N- orC-termini) or both termini or may have amino acids deleted orsubstituted. It is contemplated in this invention that a variant mayhave altered binding to a receptor than the native protein. It may haveenhanced or reduced binding which may enhance or depress a biologicalresponse. A biological response may be, for example, the stimulation ofcell division, differentiation, homeostasis or growth. A biologicalresponse may encompass other functional properties of the native proteinand would be well known to those practicing the art. Accordingly, thevariant may have altered specificity for one or more receptors. Thevariant may be generated through recombinant DNA technologies, wellknown to those skilled in the art.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe methods disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and4,965,188, hereby incorporated by reference.

Pharmacology

The present invention also contemplates pharmaceutical formulations,both for veterinary and for human medical use, which comprise as theactive agent one or more peptide(s) of the invention, as well as the useof a peptide of the invention in the manufacture of a medicament for thetreatment or prophylaxis of the skeletal conditions variously describedherein.

In such pharmaceutical and medicament formulations, the active agentpreferably is utilized together with one or more pharmaceuticallyacceptable carrier(s) therefore and optionally any other therapeuticingredients. The carrier(s) must be pharmaceutically acceptable in thesense of being compatible with the other ingredients of the formulationand not unduly deleterious to the recipient thereof. The active agent isprovided in an amount effective to achieve the desired pharmacologicaleffect, as described above, and in a quantity appropriate to achieve thedesired daily dose. The pharmaceutical composition of the presentinvention can be administered either as free forms of the peptides ofthe present invention or as pharmacologically acceptable acid additionsalts thereof.

The dose of the pharmaceutical composition of the present invention mayvary with the kind of disease, the age of patient, body weight, theseverity of disease, the route of administration, etc.; typically, itcan be administered in a daily dose of 0.5-500 microgr/kg.

Apart from other considerations, the fact that the novel activeingredients of the invention are peptides, peptide variants or fusionproteins dictates that the formulation be suitable for delivery of thesetypes of compounds. Clearly, peptides are less suitable for oraladministration due to susceptibility to digestion by gastric acids orintestinal enzymes. The peptide analogs of the present invention havebeen designed to circumvent these problems. The preferred routes ofadministration of peptides are intra-articular, intravenous,intramuscular, subcutaneous, intradermal, or intrathecal. A morepreferred route is by direct injection at or near the site of disorderor disease. Alternatively, they may be administered per orally asmicrocapsules in which the peptides of the present invention areincorporated as the active ingredient in liposome, polyamide, etc. andwhich are rendered resistant to degradation in the digestive tract.Another method of administration that can be adopted is to have the drugabsorbed through the mucous membrane such as in the rectum, within thenose or eye or beneath the tongue, so that the drug is administered as asuppository, intranasal spray, eye drop or sublingual tablet.

In one currently preferred embodiment of the present invention, cellsgenetically engineered to express high levels of an NP or an NP variantare provided. Said cells may be implanted at a suitable location, morepreferably at or near the intended site of activity, most preferably ator near an affected limb bone. In a currently more preferred embodiment,the cells are encapsulated. The encapsulated cells may be implanted at asuitable location, more preferably at or near the intended site ofactivity within the body. Implantation is preferably subcutaneous, at asite in close proximity to the growth plate of limb bones.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the peptides or peptide analogs selected from thesequences described herein, or physiologically acceptable salts orprodrugs or fusion proteins thereof, with other chemical components suchas physiologically suitable carriers and excipients. The purpose of apharmaceutical composition is to facilitate administration of a compoundto an organism.

The term “prodrug” refers to an agent, which is converted into an activedrug in vivo. Prodrugs are often useful because in some instances theymay be easier to administer than the parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility compared to theparent drug in pharmaceutical compositions.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.Pharmaceutical compositions may also include one or more additionalactive ingredients.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, grinding, pulverizing, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries. Proper formulation is dependent upon the route ofadministration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants for examplepolyethylene glycol are generally known in the art.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the variants for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from a pressurized pack or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insulator may be formulatedcontaining a powder mix of the peptide and a suitable powder base suchas lactose or starch.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active ingredients in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable natural or syntheticcarriers are well known in the art (Pillai et al, (2001) Curr Opin ChemBiol 5:447-51). Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds, toallow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forreconstitution with a suitable vehicle, e.g., sterile, pyrogen-freewater, before use.

The compounds of the present invention may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The formulations of present invention may be administered topically as agel, ointment, cream, emulsion or sustained release formulationincluding a transdermal patch. The pharmaceutical compositions hereindescribed may also comprise suitable solid of gel phase carriers orexcipients. Examples of such carriers or excipients include, but are notlimited to, calcium carbonate, calcium phosphate, various sugars,starches, cellulose derivatives, gelatin and polymers such aspolyethylene glycols.

Administration may be preferred locally by means of a direct injectionat or near the site of target or by means of a patch or subcutaneousimplant, staples or slow release formulation implanted at or near thetarget.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount of acompound effective to prevent, alleviate or ameliorate symptoms of adisease of the subject being treated. Determination of a therapeuticallyeffective amount is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

Toxicity and therapeutic efficacy of the peptides described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ (the concentrationwhich provides 50% inhibition) and the LD₅₀ (lethal dose causing deathin 50% of the tested animals) for a subject compound. The data obtainedfrom these cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient's condition(Fingl, et al. (1975) in “The Pharmacological Basis of Therapeutics”,Ch. 1 p. 1).

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved. The amount of a composition to be administered will,of course, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, and all other relevant factors.

Utilities

The present invention also relates to methods of treatment of skeletaldisorders described above, by administering to a patient in need thereofa therapeutically effective amount of the compositions of the presentinvention. The term administration as used herein encompasses oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,intrathecal, rectal, intralesional and intranasal administration.

The present invention further relates to method for the use of the NP orpeptide variants, conjugates or fusions of the present invention toprepare medicaments useful in the treatment of various skeletaldisorders such as Achondroplasia and thanatophoric dysplasia.

EXAMPLES Example 1 Ex Vivo Bone Culture

Femora derived from achondroplasia model mice (Ach369 knock-in micecarry the Gly to Cys mutation at position 369, analogous to position 375in humans or Ach374 knock-in mice that carry the Gly to Arg mutation atposition 374, analogous to position 380 in humans) were dissected fromP0 wild type, heterozygote and homozygote littermates and cultured inthe presence of natriuretic peptides for 15 days.

Protocol for bone culture: Femoral bone cultures were performed byexcising the hind limbs of mice. The limbs were carefully cleaned fromthe surrounding tissue (skin and muscles) and the femora exposed. Thefemora were removed and further cleared from tissue remains andligaments. The femora were measured for their initial length, using abinocular with an eyepiece micrometer ruler. The bones were grown in 1ml of medium (α-MEM supplemented with penicillin (100 units/ml),streptomycin (0.1 mg/ml), nystatin (12.5 units/ml), BSA (0.2%),O-glycerophosphate (1 mM) and freshly prepared ascorbic acid (50 μg/ml))with varying concentrations of NPs or CNP variants in a 24 well tissueculture dish. The bones were cultured for 15 days while measurements ofbone length and medium replacement were performed every three days. Atthe end of the experiment, the growth rate of the bones was determined.The growth rate of bones is calculated from the slope of a linearregression fit on the length measurements obtained from day 3 to 15.Units given can be converted to length, 40 units=1 mm.

Results: Ex vivo experiments with fetal bone organ culture from wildtype (normal) animals have shown that CNP, more than BNP and much morethan ANP, can induce bone elongation. According to the present inventionit is now shown that natriuretic peptides can induce longitudinal growthof Achondroplasia-derived bones. CNP increased the total longitudinalgrowth of femora derived from Ach374/+, Ach369/Ach369, Ach369/+ and wildtype (+/+ or wt) mice compared to vehicle treated animals.

FIGS. 1A and 1B depict the growth rate curves for the CNP treated Achheterozygote femora in the experiment. FIG. 2A shows the interpolationof the rates in FIGS. 1A-B. The growth rate curves shows two stages ofgrowth: an initial stage (days 0-3) in which growth is quick and is mostaffected by the presence of natriuretic peptides and a second slowerlinear rate. The maximal growth rate is observed in the initial days ofculture. This may be due to the limitations of the culturing conditions,particularly to the depletion of nutrients.

FIG. 2B shows the growth rate over 15 days in the homozygote mutant andheterozygote mutant femoras compared to wt femoras. In all cases, theCNP treated femoras increased in size at a rate higher than that of theuntreated limbs. The final length if the bone was greater in the CNPtreated groups, as well. These results suggest that Achondroplasiaderived rudiments have a high capacity to respond to CNP and are able toreach a growth rate similar to that of the wt.

Interestingly, at this stage, femora derived from homozygote Ach animalsrespond better to CNP than femora derived from heterozygote animalswhile bones derived from wt animals are very slightly influenced by CNP.This is summarized in Table 1.

TABLE 1 Growth rate of femora in CNP treated mice Growth rate Growthrate (days 0-3) (days 3-15) CNP untreated treated untreated treated +/+wild type (wt) 9 10 4.9 5.7 Ach/+ heterozygote 4.4 9.1 2.1 3.4 Ach/Achhomozygote 2.9 7.1 1.2 2.8

Example 2 Natriuretic Peptides Lead to an Increase in the Size of GrowthPlate Morphological and Immunohistological Analysis

Most of the elongation induced by natriuretic peptides observed in boneculture results from epiphyseal growth and not from elongation of themineralized diaphysis. Morphological analysis of the cellularcomposition of the growth plate in treated samples revealed that thereis an increase in the size of the proliferative region and in the sizeof the hypertrophic zone. In addition, there is an area of a cellularmatrix similar to that observed in the growth plate of FGFR3 knockoutmice. Therefore, there seems to be an increase in proliferation whichleads to a larger growth plate.

Mouse-specific BNP was synthesized and labeled with biotin. BiotinylatedBNP was tested in bone culture on bones derived from Achondroplasia miceand shown to induce bone elongation. Nevertheless, a higherconcentration of BNP was needed. Histological analysis of thedistribution of biotinylated BNP in the growth plate shows that it isapparent both in proliferative and in hypertrophic cells. Recent studieshave shown the distribution of the NP receptors in the growth plate:NPR-B is expressed in proliferative cells while NPR-C is expressed inhypertrophic cells (Yamashita et al. (2000) J. Biochem 127: 177-179).Therefore, BNP could exert its effect on bone growth either bystimulating the activity of NPR-B or by blocking the clearance of theendogenous peptide, CNP, through NPR-C.

Several signaling pathways contribute to the process of endochondralossification. The epistatic relationship between the natriuretic peptideand the FGF signaling pathways is not known. The pathways could act inparallel or one could act to induce the other. For example, if the FGFpathway were epistatic (downstream), to the NP pathway, an increase inthe activity of NP would not result in a rescue of the FGFR defects.Furthermore, if the pathways acted in parallel it would not be obviousthat an increase in NP signaling would be able to overcome the effect ofthe FGFR mutations.

Experiment 3 In vivo Administration 1. IP Administration of Drugs toAchondroplasia Pups

The fastest growth period for mice is during the first month of lifetherefore, it is expected that the earlier the experiments areinitiated, the greater the influence on growth. The disadvantage oftreating such young animals is that there is a limit on volumedelivered. Drug delivery cannot be performed intravenously (IV) or byimplantation of a continuous delivery device like a pump.

Animals: Heterozygote mice for the achondroplasia mutation, aged P4-P5,weighing approximately 3.5 gr. were randomly separated into groups of 3animals/group and ear marked. Animals were distributed 6-7individuals/cage with one foster mother.

Materials and Procedure: Animals were injected daily with drugintraperitoneally (IP) in a volume not exceeding 50 μl. This volume canbe increased proportionally to weight increase. Animals are weighed andthe tail length is measured on day 0 and every 2 days thereafter. Thetreatment is continued for 2 weeks. At the end of the administrationperiod animals are sacrificed and skeletal elements are measured andanalyzed by histology. CNP is administered at a concentration of 10⁻⁵ Mand 10⁻⁶ M (in 1×PBS) during 2 weeks.

2. Administration of Drugs to Achondroplasia Model Mice Using the AlzetPump

Drug release by osmotic pumps provides a continuous supply and aconstant amount of circulating drug. Furthermore, it enables directedrelease of the drug closer to the target site. Nevertheless, thisprocedure can only be performed in older mice.

Animals: Heterozygote mice for the achondroplasia mutation, agedP12-P14, weighing approximately 10 gr. were randomly separated intogroups of 3 animals/group and ear marked. Animals were distributed 6-7individuals/cage with one foster mother.

Materials and Procedure: The Alzet pumps used have a total volume of 100μl and a release constant of 0.25 μl/hour over a course of 14 days.Pumps were filled with appropriate drug, calibrated for 4-6 hours,implanted subcutaneously (SC) on the back of anesthetized mice and thecontents were directed with a catheter to the femoral artery of one hindlimb. Mice were monitored for recovery and returned to mothers. Micewere weighed and measured every 2 days. At the end of the administrationperiod animals were sacrificed and skeletal elements measured andanalyzed by histology. This experiment was performed with aconcentration of 10⁻⁴ M of CNP (in 1×PBS) in pump to obtain aconcentration of 10⁻⁷ M in the blood stream.

Example 4 Implantation of Alginate Encapsulated Cells that Secrete CNP

Implantation of cells expressing high levels of NPs is a further methodfor providing a continuous source of NPs. NIH-3T3 fibroblasts wereinfected with retrovirus expressing mouse CNP and selected forresistance to neomycin. CNP secretion was tested by assaying thesupernatant of the cells using RIA (RadioImmune Assay) specific for CNP(Phoenix Pharmaceuticals) and following manufacturer's instructions. Thecells are encapsulated in APA (alginate-polylysine-alginate) complexaccording to the protocol described by Chang (Chang (1997) Ann N Y AcadSci 831:461-73) and summarized below.

Materials and Methods:

1. Cell Preparation: Cells are removed from plate with trypsin,resuspended in media, transferred to 50 cc tube, spun down 5-10 minutes100 rpm, media removed.

2. PBS Wash: Cells are resuspended in PBS, spun 5-10 minutes at 1000rpm, PBS removed and cells resuspended in 0.5 ml PBS.

3. Alginate: Mix thoroughly with alginate, drawing completely in andthen out >10 times, until a homogenous mix with no bubbles is seen.

4. Encapsulate cells: Extrude alginate-cell mix into 40 ml CaCl2 1.1%solution (on ice). Start with 1/10× until spray is invisible, thenswitch to 1/100×. Watch for accumulation of alginate on end of needle:either suction off or briefly increase extrusion rate to 1/10×.Determine the exact airflow for desired capsule size by checking capsulesize/shape with check dishes. Routinely check capsules size/shapethroughout process. Approximate settings: Extrusion: 99.9 at 1/100×speed, (brief 1/10× to force out blockages) Airflow of 3-4 yields largecapsules (>600 um), 4-6 regular size capsules (generally 200-600 um),6-8 small capsules (<200 um) 7-8 very small (50-100 um) and needle: 1-2mm from end of airflow connector. Transfer capsules and CaCl2 solutionto 50 cc conical tube.

5. Capsule washes: Allow capsules to settle. Remove supernatant withsuction. Add wash solution, mix gently, and allow 2 minutes for capsulesto settle.

6. Capsule implantation: The capsules are implanted intraperitoneally(IP) into P5 mice (Ach369 and wt). The mice are weighed and tail lengthmeasured every 2 days for 3 weeks.

Example 5 Co-Administration of CNP with an NEP Inhibitor

The NPs have been shown to have a short half-life in circulation,probably due to the activity of the neutral endopeptidase (NEP). Wesought to block the activity of NEP with a specific inhibitor in orderto increase the concentration of CNP in the blood.

The experiment is performed so that CNP is administered at 10⁻⁴ M via apump to obtain a concentration of 10⁻⁷ M in blood stream together withthiorphan (NEP inhibitor) at a concentration of 10 mg/ml in pump (10μg/ml blood stream) for 2 weeks. Administration is done by using anAlzet pump as described above. The same animals are injected IP withextra CNP daily (10⁻⁷ M) together with 0.1% BSA. A similar experiment isperformed with biotinylated BNP (10⁻⁷ M, yielding 10⁻⁷ M in bloodstream) injected daily, given in conjunction with thiorphan.

In a similar manner, any combination of NPs with compounds thatcontribute to NP stability in the circulation can be administered to apatient for efficient bone elongation and/or treatment of skeletaldysplasias. Such combinations may include a mixture of CNP and BNP, NPsin combination with peptidase inhibitors or in combination with NPR-Cinhibitors. NP activity may be enhanced in combination with tyrosinekinase inhibitors.

Example 6 CNP Analogs

CNP is active as a 22 amino acid (22-mer) peptide (CNP 1-22), shown inFIG. 3 as SEQ ID NO:1. A shortened version of CNP, the 17 amino acid(17-mer) CNP 5-22 peptide, SEQ ID NO:2, was shown to be active in a cGMPassay. The 17-mer peptide comprises the amino acids of the cyclic domainof CNP and lacks five amino acids of the ectocyclic domain. NEP cleavesCNP at several sites, the primary one being the Cys6-Phe7 bond, asdepicted by the thickened arrow in FIG. 3A. Peptide analogs weresynthesized to obtain a less degradable, i.e. NEP resistant or NRP-Cresistant, peptide by incorporating one or more amino acidsubstitutions. A set of peptides including those with amino aciddeletions, insertions and substitutions is shown in Table 2. The 11 merto 15 mer peptides were shown to have reduced cGMP activity.

In some peptides, a histidine residue was incorporated between Cys6 andPhe7 (His-CNP) to disrupt the cleavage recognition site. Other peptideswere synthetically modified by methylation either on Phe7 only, or bothon Phe7 and Leu11, sites of NEP activity. Peptides may be modified byincorporation of a reporter molecule that allows detection, such as abiotin Or fluorescein, at either the N-terminus or C-terminus of thepeptide.

TABLE 2 Amino Acid Sequence of CNP Variants SEO ID NO: Amino AcidSequence % relative binding 17-mers SEQ ID NO: 2 C F G L K L D R I G S MS G L G C 127.4 SEQ ID NO: 6 C A G L K L D R I G S M S G L G C 43.8 SEQID NO: 7 C F G L K L A R I G S M S G L G C 49.1 SEQ ID NO: 8 C F G L K LD A I G S M S G L G C 18.4 SEQ ID NO: 9 C F G L K L D R A G S M S G L GC 11.5 SEQ ID NO: 10 C F G L K L D R I G S A S G L G C 138.1 15-mers SEQID NO: 11 C H F G L K L D R I G S N S - - C 25.9 SEQ ID NO: 12 C H F G LK L D R I G S M A - - C 21.7 SEQ ID NO: 13 C H F G L K L D R I G AQ S - - C 26.5 14-mers SEQ ID NO: 14 C F G L K L D R I G S M S - - - C29.9 SEQ ID NO: 15 C F G L K L D R I G A Q S - - - C 20.6 13-mers SEQ IDNO: 16 C F G L K L D R I G S M - - - - C 17.3 SEQ ID NO: 17 C F G L K LD R I G A M - - - - C 3.4 SEQ ID NO: 18 C F G L K L D R I G S Q - - - -C 11.8 SEQ ID NO: 19 C F G L K L D R I G A Q - - - - C 3.8 SEQ ID NO: 20C H F G L K L D R I G S - - - - C 11.9 12-mers SEQ ID NO: 21 C F G L K LD R I G S - - - - - C 22.7 11-mers SEQ ID NO: 22 C F G L K L D R IG - - - - - - C 4.2 SEQ ID NO: 23 C H F G L K L D R I - - - - - - C 3.8A substitution or incorporation of an amino acid is marked in bold andunderlined. Amino acid deletions are marked as a dash.

All peptide variants were analyzed for activity using the Biotrak enzymeimmunoassay (EIA, Amersham) that measures the amount of secondarymessenger, cyclic GMP (cGMP), elicited after activation of thenatriuretic peptide receptor by the peptide on C3H10T1/2 cells.Additional 17-mer variants were synthesized and tested with the resultsof the assay summarized in Table 3. Amino acid substitutions are markedin bold and underlined. The assay was performed for CNP and variants ata concentration of 10⁻⁶ M. The values are given as the percent ofrelative binding compared to CNP, where CNP 1-22 and CNP 5-22 yield 100%binding.

TABLE 3 CNP variants and in vitro cGMP values SEQ ID NO: Amino acidsequence of CNP variants % relative binding SEQ ID NO: 2 C F G L K L D RI G S M S G L G C 100.000 SEQ ID NO: 24 C A G L K L A R I G S M S G L GC −5.848 SEQ ID NO: 25 C A G L K L D R I G S A S G L G C 3.933 SEQ IDNO: 26 C F G L K L A R I G S A S G L G C 5.755 SEQ ID NO: 27 C A G L K LA R I G S A S G L G C −10.355 SEQ ID NO: 28 C I G L K L D R I G S M S GL G C 13.899 SEQ ID NO: 29 C L G L K L D R I G S M S G L G C 6.255 SEQID NO: 30 C M G L K L D R I G S M S G L G C −1.341 SEQ ID NO: 31 C W G LK L D R I G S M S G L G C −10.834 SEQ ID NO: 32 C V G L K L D R I G S MS G L G C 5.740 SEQ ID NO: 33 C H G L K L D R I G S M S G L G C 5.699SEQ ID NO: 34 C T G L K L D R I G S M S G L G C −6.903 SEQ ID NO: 35 C FG L K L E R I G S M S G L G C 9.450 SEQ ID NO: 36 C F G L K L Q R I G SM S G L G C −17.095 SEQ ID NO: 37 C F G L K L N R I G S M S G L G C17.235 SEQ ID NO: 38 C F G L K L I R I G S M S G L G C 7.228 SEQ ID NO:39 C F G L K L M R I G S M S G L G C 7.784 SEQ ID NO: 40 C F G A K L D RI G S M S G L G C 20.154 SEQ ID NO: 41 C F G I K L D R I G S M S G L G C47.395 SEQ ID NO: 42 C F G V K L D R I G S M S G L G C 64.212 SEQ ID NO:43 C F G L L L D R I G S M S G L G C 29.744 SEQ ID NO: 44 C F G L M L DR I G S M S G L G C 5.421 SEQ ID NO: 45 C F G L K A D R I G S M S G L GC 11.259 SEQ ID NO: 46 C F G L K I D R I G S M S G L G C 88.117 SEQ IDNO: 47 C F G L K V D R I G S M S G L G C 66.714 SEQ ID NO: 48 C F G L KL D H I G S M S G L G C 13.482 SEQ ID NO: 49 C F G L K L D K I G S M S GL G C 21.543 SEQ ID NO: 50 C F G L K L D Q I G S M S G L G C 11.953 SEQID NO: 51 C F G L K L D R L G S M S G L G C 14.177 SEQ ID NO: 52 C F G LK L D R V G S M S G L G C 27.103 SEQ ID NO: 53 C F G L K L D R T G S M SG L G C −1.945 SEQ ID NO: 54 C F G L K L D R I G A M S G L G C 54.469SEQ ID NO: 55 C F G L K L D R I G G M S G L G C 27.811 SEQ ID NO: 56 C FG L K L D R I G T M S G L G C 74.080 SEQ ID NO: 57 C F G L K L D R I GN M S G L G C 27.430 SEQ ID NO: 58 C F G L K L D R I G S M S A L G C53.649 SEQ ID NO: 59 C F G L K L D R I G S M S L L G C 15.289 SEQ ID NO:60 C F G L K L D R I G S M S K L G C 49.202 SEQ ID NO: 61 C F G L K L DR I G S M S G Q G C −3.474 SEQ ID NO: 62 C F G L K L D R I G S M S G M GC 51.286 SEQ ID NO: 63 C F G L K L D R I G S M S G A G C −1.111 SEQ IDNO: 64 C F G L K L D R I G S M S G G G C −10.145 SEQ ID NO: 65 C F G L KL D R I G S W S G L G C 66.450 SEQ ID NO: 66 C F G L K L D R I G S H S GL G C 66.222 SEQ ID NO: 67 C F G L K L D R I G S K S G L G C 66.910 SEQID NO: 68 C F G L K L D R I G S S S G L G C 62.531 SEQ ID NO: 69 C F G LK L D R I G S G S G L G C 62.117 SEQ ID NO: 70 C H G L K L D R I G S A SG L G C 3.058 SEQ ID NO: 71 C T G L K L D R I G S A S G L G C −9.108

The peptide of SEQ ID NO:2 was shown to be as active as the nativeCNP1-22, SEQ ID NO:1. The modification lies in the removal of theN-terminal amino acids, the ectocyclic part of the peptide. The peptideof SEQ ID NO:10 contains a substitution of Met17 to Ala. This variantretains high activity perhaps due to the alteration of an NEP cleavagesite. Furthermore, the peptide of SEQ ID NO: 10 was tested in boneculture and shown to induced elongation of Ach369 femora even at aconcentration similar to that of CNP.

The peptide variant of SEQ ID NO:6 has an amino acid substitutionwhereby Phe7 is modified to Ala. All tested substitutions of the Phe7,SEQ ID NOs: 24-25, 27-34 and 69-71, resulted in reduced activity.

Another modification includes a gain of activity obtained by thesubstitution of Met17. Met 17 is a site of NEP cleavage and itssubstitution by another amino acid including Ala identified as SEQ IDNO: 10 increases activity and may increase stability. Othersubstitutions of Met retaining activity are Trp His, Lys, Ser and Glyidentified herein as SEQ IS Nos:65-69. Other examples of variantswherein modifications were directed to the cleavage sites and activityis retained are disclosed as SEQ ID NOs: 45-47 wherein Leu 10 issubstituted, SEQ ID NOs:54-57 wherein Ser16 is substituted and SEQ IDNos:58-60 wherein Gly19 is substituted.

Peptide variants exhibiting a high level of activity as determined inthe cGMP assay may be useful for treating skeletal disorders. Thesevariants may be useful alone or in combination with other compoundsincluding but not limited to NPs, NEP inhibitors, NPR-C inhibitors or TKinhibitors. The variants with reduced activity may be useful alone or inconjugation with other NPs and may impart stability or synergy.

One skilled in the art will recognize that the variants may besynthesized as peptide mimetics. A peptide mimetic or peptidomimetic, isa molecule that mimics the biological activity of a peptide but is notcompletely peptidic in nature. Whether completely or partiallynon-peptide, peptidomimetics provide a spatial arrangement of chemicalmoieties that closely resembles the three-dimensional arrangement ofgroups in the peptide on which the peptidomimetic is based. As a resultof this similar active-site geometry, the peptidomimetic has effects onbiological systems which are similar to the biological activity of thepeptide.

Example 7 In vivo assays

Methods for detecting administered compounds in the blood or tissue oftreated mammals are known in the art. The pharmacokinetic properties ofthe administered compounds are determined using such methods. In animalmodels, radiolabelled oligonucleotides or peptides can be administeredto and their distribution within body fluids and tissues assessed byextraction of the oligonucleotides or peptides followed byautoradiography (Agrawal et al (1991) PNAS 88:7595-99). Other methodsinclude labeling of a peptide with a reporter moiety, includingfluorescent or enzyme labels, administration to an animal, extraction ofthe peptide from body fluids and organs followed by HPLC analysis.Alternatively, immunohistochemical methods are used for detection of theadministered peptide in tissue. The present invention contemplatesreporter labeled NPs and CNP variants.

Example 8 GH-CNP Fusion Construct

One approach to creating high local concentrations of a therapeuticmolecule is to target it to the growth plate (GP) via a GP-specificcarrier including but not limited to growth hormone (GH), IGF-1, TH andreceptor ligands. GH is an endocrine hormone that is secreted by thepituitary and affects bone elongation. Targeted delivery of a compoundinto cells comprising a chimeric protein has been disclosed for examplein US Patent Application No. 20010025026.

We have synthesized a GH-CNP fusion construct useful as a carrier forCNP on its way into the growth plate. This fusion protein maintains thebinding capacity of GH and the activity of CNP. In one aspect a linkerhas been added between the two peptides which enables independentfolding of each part. In another aspect, a linker comprising a cleavagesite has been introduced to allow physical separation of the twohormones. FIG. 4 shows a scheme of the constructs.

To determine whether GH can enter the growth plate from the circulation,GH was labeled with ¹²⁵Iodine and administered to mice IP, one hourafter a pulse of unlabelled GH. Blood samples were collected at severaltime points and analyzed on a protein gel. The samples show stronglabeling which increases in the first 2-3 hours, can still be observedat 6 hours and almost disappears at 24 hrs.

PCR and Cloning: Mouse Growth Hormone (mGH) and mouse CNP (mCNP) wereisolated from a brain cDNA library by method of RT-PCR, using thefollowing oligonucleotide primers (oligos):

Mouse Growth Hormone: (SEQ ID NO: 72) For: 5′ TGG CAA TGG CTA CAG ACTCTC GG (SEQ ID NO: 73 Rev: 5′ GAA GGC ACA GCT GCT TTC GAG AA Mouse CNP:(SEQ ID NO: 74 For: 5′ ACC CAA GCT TAT GCA CCT CTC CCA GC (SEQ ID NO:75) Rev: 5′ CCA TCG ATC TAA CAT CCC AGA CCG

The generated PCR products were sub-cloned into plasmids and sequencedto verify their identity and fidelity. These plasmids were then used astemplates for the generation of the fusion constructs which weregenerated by a two-step nested PCR approach. The construction isdescribed in detail here, using four different oligonucleotides andthree PCR reactions. All other constructs were generated by similarmethodology. Construct A harbors sequences encoding:

BamHI-Factor Xa Cleavage site-mCNP-Linker A-mGH-Stop Codon-Hind III.

The first PCR reaction (21 cycles) was performed on mCNP using theforward oligo which harbors sequences to encode a BamHI cloning site,Factor Xa consensus cleavage site as well as the 5′ of mouse CNP and thereverse oligo which harbors the 3′ component of CNP, as well assequences to encode linker A. The second PCR reaction (21 cycles) wasperformed on mGH using forward oligo which harbors sequences to encode alinker A as well as the 5′ of mGH and a reverse oligonucleotide whichharbors the 3′ component of mGH, including a stop codon and a HindIIIsite for subsequent sub-cloning. The third PCR reaction (21 cycles) wasperformed by combining the products of the two above PCR reactions, theforward oligo from the first PCR reaction, the reverse oligo from thesecond PCR reaction. The overlapping linker sequences for the twodifferent template DNAs, allowed the generation of the full-lengthfusions. The constructs were cloned into an expression vector and usedto transfect host cells. The fusion proteins were synthesized andisolated according to methods known in the art.

A construct wherein the CNP lies 5′ to the mGH was constructed, as well.A scheme of all the constructs that have been made is shown in FIG. 4.The legend for FIG. 4 is as follows: Linker A is a simple flexiblelinker encoding six amino acids: Gly Gly Ser Gly Gly Ser while Linker Bharbors an MMP9-specific cleavage sequence (Kridel et al. (2001) J BiolChem 276: 20572-8). B and H are BamHI and HindIII restriction enzymesites, respectively and Xa is a factor Xa cleavage site.

The activity of the GH and CNP in the fusion protein is assessed bymeasurement of cGMP accumulation for CNP and activity of GH.

Example 9 Establishment of Assay to Assess Resistance to NEP Degradation

Endothelial cells express neutral endopeptidase (NEP) in vivo and thusdegrade part of the circulating natriuretic peptides. An in vitro assayhas been established to determine the stability of the modified peptidesin the presence of endopeptidases. Endothelial cell lines have been usedbut any primary cell or cell line expressing NEP may be useful in theassay. Endothelial cell lines derived from either bovine or human tissueare grown. Cells are overlayed with 10⁻⁶ M CNP or variant, incubatedovernight and the medium is assayed for cGMP activity, as describedabove. The results obtained from the variants are compared to theactivity of CNP.

Example 10 Establishment of Assay to Assess Resistance to NPR-CDegradation

An assay to determine the resistance of the CNP variants to clearancevia the NPR-C receptor is being established using astrocytes derivedfrom a NPR-C knockout mouse (Matzukawa et al (1999) PNAS 96:7403-8).

It will be evident to the skilled artisan that administration of NPsaccording to the principles of the present invention can be performed byany suitable route of administration, utilizing any suitablepharmaceutically acceptable carrier or diluent. Under certaincircumstances specific formulations that enable or enhance targeting ofthe active principle to the bone or growth plate may be utilized such asbut not limited to use of the NPs in combination with vehicles such asliposomes, microemulsions, microcapsules, microspheres, and the like. Itis also intended antibodies, peptides, hydroxyapatite, glucosamine,collagen especially collagen type X, polyGlu or polyAsp and othermolecules having affinity for the growth plate.

It will be appreciated that many improvements may be achieved bystabilization of the NP or otherwise achieving a prolonged half life orimproved pharmacokinetic profile. For example, functional variants ofNPs having enhanced stability in vivo, such as peptides having alteredsequences or configurations, may be administered for treatment ofskeletal dysplasias and/or bone elongation.

Further, cells can be engineered to produce and secrete functionalvariants of NPs having enhanced stability in vivo and these cells may beencapsulated in APA (alginate-polylysine-alginate) complex and implantedintraperitoneally for treatment of skeletal dysplasias and/or boneelongation.

While the present invention has been particularly described, personsskilled in the art will appreciate that many variations andmodifications can be made. Therefore, the invention is not to beconstrued as restricted to the particularly described embodiments,rather the scope, spirit and concept of the invention will be morereadily understood by reference to the claims which follow.

1. A method for the treatment of skeletal dysplasia comprising the stepof administering to a patient a composition comprising a therapeuticallyeffective amount of at least one natriuretic peptide.
 2. The methodaccording to claim 1, wherein the at least one natriuretic peptide is aC-type natriuretic peptide (CNP).
 3. The method according to claim 2,wherein the C-type natriuretic peptide is human CNP as set forth in SEQID NO:
 1. 4. The method according to claim 1, wherein the compositionfurther comprises a pharmaceutically acceptable carrier or excipient. 5.The method according to claim 4, wherein the pharmaceutically acceptablecarrier or excipient is selected from polymers, calcium carbonate,calcium phosphate, various sugars, starches, cellulose derivatives, andgelatin.
 6. The method according to claim 4, wherein thepharmaceutically acceptable carrier or excipient is polyethylene glycol.7. The method according to claim 1, wherein the composition comprisesnatriuretic peptide secreting cells.
 8. The method according to claim 7,wherein the natriuretic peptide secreting cells are transplanted orimplanted to the site of a lesion.
 9. The method according to claim 7,wherein the natriuretic peptide secreting cells are encapsulated withinan inert matrix.
 10. The method according to claim 9, wherein thenatriuretic peptide secreting cells are encapsulated within analginate-polylysine-alginate complex.
 11. The method according to claim1, wherein the composition is administered in liquid or suspension form.12. The method according to claim 1, wherein the composition isadministered in an injectable, implantable or transplantable form. 13.The method according to claim 1, wherein the skeletal dysplasia isachondroplasia.
 14. The method according to claim 1, wherein thetreatment comprises abnormal bone growth.
 15. The method according toclaim 14, wherein bone growth comprises bone elongation.